Prospect of acromegaly therapy: molecular mechanism of clinical drugs octreotide and paltusotine

Somatostatin receptor 2 (SSTR2) is highly expressed in neuroendocrine tumors and represents as a therapeutic target. Several peptide analogs mimicking the endogenous ligand somatostatin are available for clinical use, but poor therapeutic effects occur in a subset of patients, which may be correlated with subtype selectivity or cell surface expression. Here, we clarify the signal bias profiles of the first-generation peptide drug octreotide and a new-generation small molecule paltusotine by evaluating their pharmacological characteristics. We then perform cryo-electron microscopy analysis of SSTR2-Gi complexes to determine how the drugs activate SSTR2 in a selective manner. In this work, we decipher the mechanism of ligand recognition, subtype selectivity and signal bias property of SSTR2 sensing octreotide and paltusotine, which may aid in designing therapeutic drugs with specific pharmacological profiles against neuroendocrine tumors.


Seen from the
, the octreotide also showed higher affinity and efficacy on SSTR2 than other subtypes of SSTR family members, is there any explanation on it based on the structure? 3. For the selectivity analysis, some comparison between paltusotine and the small molecule L-054,264 might be helpful. 4. For the signal bias part, the relationship between microswitch residues and signal bias with different ligands were not clarified. Is the response to signal bias ligand dependent? 5. What's the influence of the microswitch residues on the receptor internalization for both ligands? 6. Since the authors repetitively emphasized the subtype selectivity and biased signaling, I'm not clear on what pharmacological profile is therapeutically beneficial for SSTR2 drug discovery? (subtype selectivity? Signaling bias?) is there any clinical evidence? 7. Some structural analysis does not seem reliable, for example, Line 218-219: "Superposition of the active state of SSTR2 with SSTR3 (predicted active model from GPCRdb)…" this is a comparison between an experimental structure for protein A and predicted model for protein B, then why we need experimental structure? 8. For many side chain analylsis, it is in the context of a ~3.3Å resolution cryo-EM map, and the side chain placement, at least for some of them, could be ambiguous. Therefore, for some key residues (engaging core interactions with the two ligands) on the receptor, density map should be shown.
Minor comments: 1. The "Discussion" part looks more like a "Summary", which should be further revised. Especially, the authors suggested future studies on inactive SSTR2 structure which has been recently reported by Skiniotis, G. et al (PDB 7UL5). 2. There are some typos and grammatical errors which need careful check. For example, Line 215, by "combing with …" should be combining.
Reviewer #2: Remarks to the Author: In this manuscript, Zhao, J. et al report the cryo-EM structures of the human somatostatin receptor 2 (SSTR2) -Gi complexes bound with either the first-generation peptide drug octreotide or the newgeneration small molecule drug paltusotine. The structures reveal the molecular mechanism of the subtype selectivity of paltusotine towards the SSTR2 over other SSTR subtypes. What is more interesting, paltusotine exhibits better G-protein biased signaling compared to octreotide and exhibits a potential better performance in promoting apoptosis of pituitary tumor cells.
Due to the importance of SSTR2 as drug target, many groups have solved the structures of SSTR2 bound with different ligands. I found at least five publications through a quick search: However, the authors only cited two of them in this manuscript. I think all of these five papers should be cited and discussed. For example the NMSB paper also reports the SSTR2-octreotide structure, but with a higher resolution (2.7Å vs 3.37Å). The readers would be interested to know if the structure models are similar or different in these two works. Furthermore, the authors claim that "Future studies would be highly informative to solve the inactive SSTR2 and ……" (line 333) while the inactive SSTR2 structure has been solved and reported in the Cell research paper by Zhao, W. et al.
The highlight of this work, in my mind, is the studies of β-arrestin recruitment and internalization. However, I am not totally convinced that N2766.55 and F2947.35 play key roles to the signal bias of SSTR2 when sensing octreotide. The N2766.55A and F2947.35A mutation indeed cause larger reduction in octreotide stimulated β-arrestin recruitment compared to Gi signaling, but I am not sure if the differences are due to biased signaling or just due to impaired affinity. For example, N2766.55A causes ~90 fold increase in EC50 for octreotide stimulated Gi activation and ~540 fold increase for βarrestin recruitment. It's possible that this mutation just reduced the affinity of octreotide and made this ligand less functional in activating SSTR2. This affinity reduction has a larger effect on β-arrestin recruitment assay because β-arrestin generally binds weaker to the receptor than G protein. I also noticed that the current β-arrestin recruitment assays were done without co-transfection of GRK. It would be more convincing if the authors could repeat the assay in presence of GRK and check if these two mutations indeed abolish β-arrestin recruitment.
One interesting question is why paltusotine is more G protein biased compared to octreotide, as this information may guide future drug design. But the authors did not really address this question.
Other minor suggestions include: 1. In line 29, the authors claim that "drug resistance occurs in a subset of patients, which may be correlated with SSTR subtype selectivity or cell surface expression." I understand the correlation between 'drug resistance' and 'cell surface expression'. But I don't quite understand why 'subtype selectivity' correlates with 'drug resistance'. 2. The confocal fluorescence microscopy images are confusing to me. For example, in Fig.6c and 6d, it looks like paltusotine induces as much internalization as octreotide does in WT SSTR2. While in Fig.  6c, it looks like octreotide induces as much internalization for N2766.55A and F2947.35A mutants compared to WT SSTR2. These results are not consistent with the main conclusion of the manuscript.
We thank the referees for their valuable time in reviewing our manuscript and the constructive suggestions that they have provided. Please find our responses to the specific comments raised by the reviewers below. We have copied each comment in Italic, which is followed by our own point-by-point response in blue, including details about the corresponding changes to the manuscript.

Reviewer #1:
In this manuscript, the authors reported molecular mechanism of octreotide and paltusotine for SSTR2. Additionally, the structural and mutagenesis assay provided some insight to the subtype-selectivity and biased signaling mechanism for SSTR2. Overall, I think the structures and functional analysis have some values to the SSTR2 pharmacology. However, the broad impact to the GPCR research field in regards to the subtype selectivity and biased signaling mechanism are lacking.
Response: Thank you so much for taking the time to evaluate our work. We appreciate your constructive comments that improved our study. In the revised manuscript, we have carried out additional experiments and included the results about the mechanisms of ligand selectivity and bias signal. Response: We thank for the reviewer's insightful comments. Previous studies mentioned by reviewer described about the mechanism of ligand recognition, receptor activation, as well as subtype selectivity of the group 2 (SSTR2/3/5) receptors vs. group 1 (SSTR1/4). SSTR2 signals via activation of Gi protein and engages β-arrestin to mediate distinct cellular signaling events, however, pharmacological properties of different types of ligands (peptide octreotide and small molecule paltusotine) remain unclear. Here, one of the new insights in our work is that we first characterized the pharmacological profiles of octreotide and paltusotine at SSTR2. On the other hand, our study determined the selective mechanism of paltusotine for SSTR2 among group 2 SSTRs (SSTR2/3/5). Similar with other GPCR subtypes, SSTRs distributed in different tissues and regulated divergent physiological processes. Therefore, designing selective ligands that can achieve receptor subtype selectivity and specific receptor signaling and even control on-or off-target side effects, could be safer therapeutic strategy in GPCR drug discovery field.
Compared with the published studies about SSTR2, we supplied the mechanisms of bias signaling and SSTRs group 2 subtype selectivity in this manuscript. In addition, by performing cell-based G-protein activation assay, β-arrestin recruitment assay, and receptor internalization analysis, we demonstrated pharmacological features of different generations of drugs targeting SSTR2. Furthermore, we measured the pituitary tumor cell GH3 apoptosis after administration of octreotide and paltusotine by using flow cytometry. In general, our finding provides comprehensive insights into understanding the structural basis of SSTR2 and the functional actions of divergent drugs.
(c) while I appreciate the independent work of this manuscript, a through comparison between different ligand binding modes, and the comparison of the same ligand-bound structures (to validate structural reliability by independent methods) would be needed.
Response: Thanks for the valuable comment. We do agree that the comparison of the structures solved in this study with those in the previous studies are significative and conclusive. In the revised manuscript, i) we first carried out structural comparison of peptide ligands binding modes in SSTR2, including agonist octreotide and endogenous agonist SST14. ii) The detailed ligand recognition and the critical microswitches required for receptor activation were further analyzed in the different states of the receptor. iii) In addition, we compared the binding mode of small molecules in SSTR2.
The related descriptions are presented below and the related discussion has been included in the result section 2 "Recognition mechanism of octreotide by SSTR2" (lines 124-128, 134-135, 140-143) and the discussion section. As is shown in Table R1, we summarized the reported structures of SSTR2 so far. The structures of octreotide-bound SSTR2 have been determined by Skiniotis and Tian groups (PDB ID: 7T11 and 7XAU), structural comparisons of SSTR2-Gi in complex with octreotide with the previous two signaling complexes reveal the nearly identical assembly architecture ( Fig. R1a-d), with a RMSD of 0.82 -1.07 Å for the Cα atoms of the receptor. Additionally, the binding of SSTR2 with octreotide in these three complex structures exhibit the same recognition mechanism, despite different Gi proteins (Gi1 or Gi3) couple.
Both approved drugs octreotide and lanreotide contain pharmacological core region ((D)-Tyr4 and Lys5) that is necessary for receptor activation and inserts into the bottom of the orthosteric pocket, subsequently triggering the rearrangement of microswitches for receptor activation (Fig. R1e, f), for instance, the toggle switch W 6.48 , PIF and DRY motifs, as well as NPxxY motif. These key residues or motifs in the structures of octreotide-or lanreotide-bound SSTR2 exhibit similar conformation as endogenous peptide SST14-bound structure. Moreover, two structures of small-molecules L-054,522-and L-054,264-bound SSTR2 were reported by Mao and Zhao groups, respectively. It is noteworthy that paltusotine displays different scaffolds from L-054,522 and L-054,264. Structural comparison of paltusotine-SSTR2 structure with these two structures reveal a common binding site as well as extended binding sites for specific moieties (Fig. R1g). All of the three molecules occupy the core binding region, L-054,264 and L-054,522 share the extended binding region, whereas paltusotine occupies a minor pocket. In detail, the conformation of the residues involved in the core binding region of the three SSTR2 structures are almost identical (D122 3.32 , Q126 3.36 , F208 5.38 and Y302 7.43 ) (Fig. R1h). The large 3,5-difluorophenyl moiety of paltusotine is placed toward the extracellular end of TM2, creating the minor pocket formed by Y50 1.39 , L99 2.60 , Q102 2.63 , V103 2.64 and D295 7.36 in SSTR2 (Fig. R1h). While L-054,264 places into the extended binding region formed by TM6 and TM7, and forms extensive interactions with F275 6.54 , N276 6.55 , S279 6.58 , L290 7.31 and F294 7.35 , which are absent in paltusotine binding (Fig.   R1h). As for L-054,522, it is much larger than paltusotine and L-054,264 in size. It also interacts with the paltusotine binding minor pocket (L99 2.60 , Q102 2.63 , V103 2.64 and D295 7.36 ), on the other hand, L-054,522 extends into TM6, TM7 and ECL3 like L-054,264, and forms extensive interactions with F275 6.54 , N276 6.55 , L290 7.31 and P286 ECL3 (Fig. R1i). L-054,522 is just like the chimera of L-054, 264 and paltusotine. in this study. a. Overall structural comparison of SSTR2-octreotide in our study with SSTR2 7T11 . SSTR2 in our study is shown as cartoon and colored in green-cyan, octreotide is shown as sticks and colored in light pink; SSTR2 in SSTR2 7T11 is shown as cartoon and colored in gray, octreotide is shown as sticks and colored in yellow. b. The binding poses of octreotide in the two structures. c. Structural comparison of the residues in the octreotide binding pocket of SSTR2 in the two structures. Key residues in SSTR2 are shown as sticks. d. Conformational comparison of the microswitch residues of SSTR2 in these two structures. Key residues are shown as sticks. (see also in supplementary Fig. 4b-c) e: Structural alignment of SSTR2-octreotide structure solved by this study (colored in green-cyan) with the inactive SSTR2 structure (PDB code: 7UL5, colored in wheat). Microswitch residues are shown as sticks. (see also in supplementary Fig. 4a) f: Structural alignment of SSTR2-octreotide structure solved by this study (colored in green-cyan) with the lanreotide-bound (PDB code: 7XAV, colored in gray) or SST14-bound SSTR2 structure (PDB code: 7T10, colored in wheat). Microswitch residues are shown as sticks. in SSTR2-L-054,522 7XN9 is shown as cartoon and colored in wheat, L-054,522 is shown as sticks and colored in yellow. Key residues involved in ligand binding in SSTR2 are shown as sticks, residues crucial for both ligands are labeled in black; residues only crucial for paltusotine binding are labeled in violet; residues only involved in L-054,522 binding are labeled in yellow. Supplementary Fig. 5a, the octreotide also showed higher affinity and efficacy on SSTR2 than other subtypes of SSTR family members, is there any explanation on it based on the structure? Response: Thank you for the question. Indeed, our data reveal that octreotide showed higher efficacy on SSTR2 (SSTR2 >SSTR3/5 > SSTR1/4), which is consistent with the previous study 1 . In the revised manuscript, we analyzed the possible reason why octreotide exhibited higher efficacy than other subtypes of SSTR members, and then highlighted some points based on the structural comparison as well as mutagenesis studies.

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First of all, combined with the previously published papers of SSTR2 2,3 as well as sequence alignment of SSTRs, the residues F 7.35 , N 6.55 , and Q 2.63 were found to participate in subtype selectivity of octreotide on SSTR2 over group 1 SSTRs (SSTR1/4), which were conserved among SSTR2, R3 and R5. Accordingly, mutating these three residues in SSTR2 to the corresponding residues in SSTR1 (F 7.35 S and N 6.55 Q, Q 2.63 S) markedly reduced the octreotide binding efficacy of SSTR2.
Consistently, as was shown in our structure, F294 7.35 S substitution probably abolish the hydrophobic interactions, whereas N276 6.55 that is close to Trp4 in octreotide and N276 6.55 Q replacement could result in steric clash with octreotide ( Fig. R2a). In addition, Q102 2.63 is observed to make direct interaction with Thr6 in octreotide-bound SSTR2 structure (Fig. R2a), while the equivalent residue S 2.63 in SSTR1 and R4 could disrupt such contact and weaken the activation efficacy of octreotide. In a word, these residues contributing to the stable binding of octreotide with group 2 SSTRs (SSTR2/3/5) over group 1 SSTRs (SSTR1/4). Secondly, octreotide showed moderate affinity for SSTR3/5 over SSTR2. To clarify the subtype selectivity of octreotide within group 2 SSTRs (SSTR2/3/5), we combined the sequence alignment with structural analysis (Fig. R2a, b) and focused on the non-conserved residues within the octreotide binding pocket. As was shown in our structure, T194 45.51 interacts with Thr6 in octreotide, and it was replaced by histidine or asparagine in SSTR3 or R5, which might be responsible for the reduced octreotide binding affinity of SSTR3/5. Our cAMP inhibition assays showed that T194 45.51 H substitution in SSTR2 (Fig. R2c), which may impair the contact between octreotide and SSTR2, significantly reduced the receptor activation.
In the revised manuscript, we have included related description in lines 375-378.   (Fig. R3a). Consistent with previous reporting 4 , our results of cAMP inhibition measurement revealed that both L-054,264 and paltusotine behave more potent selectivity for SSTR2 relative to other subtypes (Fig. R3b, c). In the study published by Mao group discussed the selective mechanism of L-054,264 on SSTR2, and they indicated that the residues F275 6.54 , F294 7.35 and N276 6.55 determined the selective recognition of L-054,264 by SSTR2 5 , however, paltusotine lacks the direct contacts with these residues due to different binding pose in SSTR2. Furthermore, we tended to focus on the minor binding site engaged in paltusotine binding. We next generated substitution of the nonconserved residues from sequence alignment (Fig. R3d), and the results of cAMP inhibition measurement revealed that replacement of V103 2.64 and T194 45.51 with the corresponding residues (N103 2.64 and H194 45.51 ) in SSTR3 reduced the activation of SSTR2 induced by paltusotine (Fig. R3e), meanwhile, both N101 2.64 V and H192 45.51 T substitutions in SSTR3 increased the receptor activation (Fig. R3f). Thus, our results indicated that V103 2.64 and T194 45.51 involved in SSTR2 selectivity when sensing paltusotine. These findings indicated the multifaceted subtype selective mechanism.
The related descriptions have been included in the result section 4 "Selectivity of paltusotine for SSTR subtypes", lines 223-235.

For the signal bias part, the relationship between microswitch residues and signal bias with different ligands were not clarified. Is the response to signal bias ligand dependent?
Response: Thanks for the valuable comment. According to the suggestion, we further investigated the contribution of the key microswitches of SSTR2 to the signal bias property, including toggle switch W 6.48 , PIF, DRY and NPxxY motifs. The result of our cAMP inhibition and β-arrestin recruitment assays indicated that alanine substitution of key residues from those microswitches significantly reduced the potency of βarrestin recruitment, but only slightly influenced the G-protein activition induced by octreotide ( Fig. R4a-f). Particularly, in agreement with octreotide stimuli, those mutants of SSTR2 exhibited similar manner in G-protein activation or β-arrestin recruitment in response to paltusotine (Fig. R4g-l). Collectively, our finding demonstrated that critical microswitches contribute to the β-arrestin signal bias of SSTR2, the relationship between microswitch residues with signal bias of GPCR have been investigated in previous studies [6][7][8] . Meanwhile, the bias property of SSTR2 microswitch residues were similar when sensing both octreotide and paltusotine, which indicates that the microswitch residues involved signal bias property is not ligand dependent. The related descriptions have been included in the result section 5 "Signal bias properties of SSTR2 with different ligands", lines 310-314.

What's the influence of the microswitch residues on the receptor internalization for both ligands?
Response: We thank the reviewer for pointing out detecting the influence of the microswitch residues on the receptor internalization. According to the suggestion, we explored the impact of the microswitch residues on SSTR2 internalization by performing ELISA assay and receptor trafficking by Bystander BRET assays, with cotransfecting with FYVE, an early endosome sensor 9,10 . Consistent with our β-arrestin recruitment assays, alanine substitution of microswitch residues resulted in reduced receptor internalization when sensing both octreotide (Fig. R5a, b) and paltusotine (Fig.  R5c, d). Therefore, our data suggests that the internalization of SSTR2 can be affected by microswitch residues. The related descriptions have been included in the result section 6 "Influence of SSTR2 internalization by the ligand-induced β-arrestin signal", lines 324-331.   15 . Further, an experimental study from over 30 acromegalic patients revealed that the expression level of β-arrestin 1 had an crucial role in the modulation of SST analogs drug efficacy as well as growth hormone secretion, which suggests that lower expression of β-arrestin 1 in pituitary adenomas may be associated with decreased recycling rate of SSTR2 and better SST analog response 16,17 . It is noteworthy that a recent reporting from Crinetics Pharmaceuticals, Inc. and University of Texas Health Science center suggested that internalization might be thought to limit the therapeutic effect of SST analogs, and they announced a small molecule named paltusotine (under clinical phase 3 currently) with improved efficacy by activating Gibiased signaling and reducing desensitization and internalization of SSTR2 18 .
Collectively, together with previous literatures, we provide insights to understand the correlation of subtype selectivity and signaling bias of SSTR2 with therapeutic efficacy.
The related descriptions have been included in the "introduction" section paragraph 2 and 3, lines 63-65 and 74-79.

Some structural analysis does not seem reliable, for example, Line 218-219: "Superposition of the active state of SSTR2 with SSTR3 (predicted active model from GPCRdb)" this is a comparison between an experimental structure for protein A and predicted model for protein B, then why we need experimental structure?
Response: Thanks for your helpful suggestion. We apologize about the confusing description in the manuscript. In the revised manuscript, we aim to examine the subtype selectivity of paltusotine for distinguishing group 2 SSTRs (SSTR2, R3 and R5), so the accurate three-dimensional structure of SSTR2 determined by experimental technique is being pursued. Structural visualization of paltusotine-bound SSTR2 complex can confirm the interaction mode of ligand in the orthosteric site of SSTR2, and sequence alignment among SSTRs could provide an opportunity for us to discuss the differences.
Furthermore, by carefully analyzing the binding model of paltusotine and octreotide with SSTR2, we eventually found 6 residues that were not conserved among group 2 SSTRs with the ligand binding pocket. We next examined the role of divergent residues in receptor activation by performing residue substitution and measuring cAMP inhibition efficacy, hopefully, the predicted model of SSTR3 based on homologous structure could aid in understanding the results of cell-based assays as well as the mechanism of subtype selectivity of paltusotine for SSTR2 among group 2 SSTRs. To avoid this confusion, we moved the related main figure to supplementary files in the revised manuscript.
8. For many side chain analysis, it is in the context of a ~3.3Å resolution cryo-EM map, and the side chain placement, at least for some of them, could be ambiguous. Therefore, for some key residues (engaging core interactions with the two ligands) on the receptor, density map should be shown. Response: Thanks for the reviewer's helpful suggestion. In the revised manuscript, we have shown the density maps of the side chain of key residues engaged in ligand recognition, further supporting the believable modes of ligand-bound SSTR2 (Fig. R6).
In addition, we included the figure of the density maps in the Supplementary Fig. 3. Response: Thanks for the constructive suggestion. In the revised manuscript, we have included structural comparison of active with inactive state, receptor activation, and subtype selectivity as well as biased therapeutic of SSTR in the revised discussion section. Our findings provide insights into understanding the safe window of therapeutic agents with biased pharmacology in GPCR drug discovery field.
We first checked the inactive structure of SSTR2 without ligand binding (PDB code: 7UL5) and the antagonist peptide CYN 154806-bound inactive structure of SSTR2 (PDB code: 7XNA). As suggested, the structural comparison of our structures with these two inactive structures reveals that the agonist paltusotine inserts in the core binding region of SSTR2 deeply, forming direct interaction with the side chain of Q126 3.36 , simultaneously, the conformational displacement of Q126 3.36 may cause rearrangement of the microswitches such as W269 6.48 , I130 3.40 and F265 6.44 in PIF motif, finally altering the conformation of TM5 and TM6 to achieve G-protein coupling (Fig.  R7).
Similar with other GPCR subtypes, SSTRs are distributed in different tissues, regulating divergent physiological processes. SSTR2 is a valuable drug target for the treatment of many diseases such as acromegaly. The high sequence homology among SSTR subtypes and divergent bias profiles of SSTR2 call for the development of therapeutic drugs toward specific subtypes and signaling pathways. Here, we characterized the pharmacological profiles of the clinical drug paltusotine and the firstgeneration drug octreotide. Structural determination of paltusotine-bound and octreotide-bound SSTR2-Gi signaling complexes elucidate the molecular mechanism of the recognition of paltusotine and octreotide by SSTR2. During preparation of our manuscript, several individual groups reported the structures of SSTR2 bound to octreotide and other different types of ligands 2,3,5,19,20 . We noticed that the octreotidebound structures from three independent groups exhibit nearly identical conformation.
In addition, these studies profiled the subtype selectivity of ligand to group 2 SSTRs (SSTR2/3/5) over group 1 SSTRs (SSTR1/4). Herein, we further investigated the mechanism that paltusotine discriminates specific subtype from group 2 SSTRs via an unusual minor pocket in SSTR2. Emerging of the structures of other SSTRs will be helpful to understand selective mechanism and could provide more information to design selective small molecules toward specific SSTR subtype.
Previous clinical data showed that the expression level of SSTR2 from acromegalic patients occurred down-regulated 15 during the administration of SST analog drug octreotide, and the expression level of β-arrestin 1 in over 30 acromegalic patients were demonstrated to be important for modulation of the efficacy of SST analog drugs as well as growth hormone secretion, which suggests that lower expression of β-arrestin 1 in pituitary adenomas was associated with decreasing recycling rate of SSTR2 and better SST analog response 16,17 . The result of our functional assay revealed that paltusotine displayed more G protein-biased property compared with octreotide. By further inspecting the differences between octreotideand paltusotine-induced SSTR2 biased signaling, we found that paltusotine loses the interactions with I284 ECL3 , K291 7.32 , N276 6.55 and F294 7.35 , which participated in the recruitment of β-arrestin induced by octreotide. Our study, to a certain extent, contributes to the understanding of the functional bias of ligands and guiding rational drug design targeting SSTRs. Thus, designing selective ligands that can achieve receptor subtype selectivity or specific receptor signaling and even control on-or offtarget side effects, could be safer therapeutic agents in GPCR drug discovery field.

There are some typos and grammatical errors which need careful check. For example, Line 215, by "combing with …" should be combining.
Response: We thank the reviewer for pointing out this issue. We have checked through the whole text carefully and corrected some typos and grammatical errors in the revised manuscript.

Reviewer #2:
In this manuscript, Zhao, J. et  Response: We thank the reviewer for taking the time to evaluate our work. We do agree that it is of importance to compare available structures of SSTR2 and analyze the similarities and differences among them. According to the helpful suggestion, we first summarized the published complex structures of SSTR2, and we also included structural perspectives of SSTR2 in response to different types of ligands in the result and discussion sections in the revised manuscript, lines 124-128, 134-143. Compared with SSTR2-octreotide complex structure at a higher resolution of 2.7 Å reported by Skiniotis group (PDB code: 7T11), the SSTR2-octreotide structure determined here displays nearly identical conformation with a RMSD of 0.82 Å for the Cα atoms of the receptor (Fig. R8a). In detail, the two octreotide molecules fold the same pose in both structures (Fig. R8b, c), even though SSTR2 couples different Gi proteins (Gi1 or Gi3). Further structural comparison reveals the same recognition manner, in which the key residues (D)-Trp4 and Lys5 of octreotide locate at the bottom of the orthosteric pocket, further triggering extracellular signal transmembrane transduction. The critical microswitches required for receptor activation, including the toggle switch, PIF and DRY motifs, are observed to exhibit the identical conformation upon Gi protein coupling (Fig. R8d). Taken together, the structures of SSTR2-Gi complex bound to octreotide determined by different groups all represented the active signaling complex, there is no significant difference among them except for the extracellular tips of the complex structure due to the dynamic features of the receptor.
The available structures basically provide us opportunities to investigate the mechanism of ligand recognition and receptor activation.
Moreover, as the reviewer mentioned, an antagonist-bound inactive structure of SSTR2 has been reported by Zhao group (PDB code: 7XNA, Cell research), as well as an inactive structure of SSTR2 without ligand has been reported by Skiniotis group (PDB code: 7UL5). The antagonist CYN 154806 contains the key residues (D)-Trp8-Lys9-Thr10-Cys11 that is also present in octreotide (in octreotide it is numbered in (D)-Trp4-Lys5-Thr6-Cys7). Subsequently, we compared the inactive state of SSTR2 bound to antagonist CYN 154806 with the active state of SSTR2. Structural comparison reveals that Phe5-(D)-Cys6 of CYN 154806 folds into distinct pose from the corresponding (D)-Phe1-Cys2 in octreotide. More importantly, (D)-Trp4 in octreotide or the hydroxybenzonitrile moiety in paltusotine, as the key facets for receptor activation, inserts more deeply than the antagonist CYN 154806, further stabilizing the extracellular regions by packing with TM bundle in the activated SSTR2 structure. In contrast, the equivalent residue (D)-Trp8 in CYN 154806 is tilted and inserts into a different hydrophobic site, thus losing contact with TM6. In addition, CYN 154806 is observed to occupy another extended binding pocket (EBP-2) at the extracellular portion, such interaction of EBP-2 with antagonist might hinder the narrowing of the extracellular regions of SSTR2 for activation (Fig. R8e).
In all, we have added the comparison of these structures in the discussion section, and analyzed the similarities and divergencies within these structures. These five papers mentioned above have been cited in the revised manuscript. a: Overall structural comparison of SSTR2-octreotide in our study with SSTR2 7T11 . SSTR2 in our study is shown as cartoon and colored in green-cyan, octreotide is shown as sticks and colored in light pink; SSTR2 in SSTR2 7T11 is shown as cartoon and colored in gray, octreotide is shown as sticks and colored in yellow. (see also in Supplementary Fig. 4b) b: The structure models of octreotide in these two structures.  Response: We thank the reviewer's meaningful question and advice. We totally understand the reviewer's concern. Previous ligand binding assays of SSTR2 were taken in competition with 125 I-SST14. Unfortunately, we could not get this radioligand due to the long shipping time from abroad during the COVID-19 pandemic.
We applied intramolecular fluorescent arsenical hairpin bioluminescence resonance energy transfer (FlAsH-BRET) method instead to monitor the conformation changes of SSTR2 in response to different types of ligands, especially the extracellular regions occur notable rearrangement upon ligands binding to the orthosteric site 21 , and the results of the measurement can reflect the ligand binding ability with the receptor at a certain degree 22,23 .
We therefore designed five sites at three extracellular loops (ECL) for incorporating with FlAsH motif (Fig. R9a), and the sensor Nluc was introduced to the N-terminus of SSTR2. The BRET signal between Nluc-N terminus and FlAsH-ECL exhibited a notable increase at position I284 ECL3 labelling (Fig. R9b). Then, we measured the BRET signals at two different time points after ligand administration. Compared with wild-type SSTR2, the results of 3 min administration from N276 6.55 A or F294 7.35 A mutant revealed that the binding of octreotide or paltusotine to the receptor exhibited similar conformation changes, which means that the mutations at position 6.55 and 7.35 did not affect the recognition of octreotide or paltusotine by SSTR2 (Fig.  R9c, e). Whereas the results of 9 min administration indicated both mutants decreased the BRET signals when sensing octreotide, by contrast, N276 6.55 A or F294 7.35 A mutation of SSTR2 retained similar signals with wild-type SSTR2 in response to paltusotine (Fig. R9d, f). Collectively, N276 6.55 A or F294 7.35 A mutation may influence the conformation of SSTR2 in response to different ligands. Consistent with our finding, the results of the competition assays from Zhao group 20 reveals that F294 7.35 A mutation reduced 6-fold binding affinity relative to wild-type SSTR2. In our β-arrestin recruitment assays, we measured the signal after 3 min ligand administration, the results suggested that N276 6.55 A and F294 7.35 A mutations were associated with signal bias of SSTR2. Of course, we can't exclude the possibility that these mutations could affect the binding affinity with the receptor.
Meanwhile, we agree with the reviewer's opinion, the affinity reduction might have effect more on the β-arrestin recruitment assays compared with the G protein signaling. We wondered whether a key residue mutant from SSTR2 could affect the binding affinity and G protein signaling but still retain similar ability to recruit βarrestin compared to wild-type receptor. It is noteworthy that a mutation Y302 7.43 A of SSTR2 in the orthosteric site slightly impaired the octreotide induced β-arrestin recruitment, however, the Y302 7.43 A substitution nearly abolished the binding of octreotide to SSTR2 20 . Given that the effector β-arrestin coupling to the intracellular portion of SSTR2 is likely to promote the receptor to recognize the extracellular orthosteric ligands in an allosteric manner, mutating the residue that only related to the ligand binding may not affect β-arrestin recruitment in some extent (especially affect β-arrestin recruitment more than G protein signaling). In a word, our results suggest that N276 6.55 and F294 7.35 should be related with the β-arrestin signal bias of SSTR2 by octreotide ( Fig. R9g-j). We have modified the related description and included the FlAsH-BRET results in the revised manuscript in the section of Result 5 "Signal bias properties of SSTR2 with different ligands" section, lines 286-294.  Response: We thank the reviewer's constructive suggestion. Previous study has shown that β-arrestin recruitment by SSTR2 was associated with GRK2 and GRK3, which belonged to the same GRK branch 24,25 . Therefore, we repeated the β-arrestin recruitment assays with co-transfection of GRK2 according to the suggestion.
Consistently, we did not observe significant differences for the efficacies of ligand induced β-arrestin recruitment in the presence or absence of GRK2. For instance, both N276 6.55 A and F294 7.35 A mutants of SSTR2 nearly abolished the ability to recruit cellular β-arrestin in response to octreotide, by contrast, these two mutants slightly influenced paltusotine induced β-arrestin recruitment (Fig. R10). We have included the related description in Result 5 "Signal bias properties of SSTR2 with different ligands" section, lines 283-285. One interesting question is why paltusotine is more G protein biased compared to octreotide, as this information may guide future drug design. But the authors did not really address this question. Response: Thanks for the valuable comment. As discussed in our study, the small molecule paltusotine induced lower β-arrestin recruitment relative to octreotide and exerted as a G protein-biased ligand ( Supplementary Fig. 8a). In the revised manuscript, we have provided descriptions to address the relationship of signal bias of SSTR2 with future drug design, and further modified the section of "Signal bias properties of SSTR2 with different ligands''. Briefly, we first compared the two SSTR2 structures in complex with octreotide and paltusotine and carefully analyzed the key residues engaged in ligand recognition. As is shown in Fig. 3b (main figure), we noticed that octreotide occupied an extended binding pocket of SSTR2, subsequently, by generating a range of mutations in the extended binding pocket, we found that alanine substitution of I284 ECL3 , K291 7.32 , N276 6.55 residues resulted in markedly reduced β-arrestin signal (Fig. R11a, c), but only slightly affected the Gi pathway induced by octreotide (Fig. R11b, c). By contrast, the small molecule paltusotine only occupies the core region of orthosteric site in SSTR2, losing the interaction manner that is shown in octreotide binding, displaying lower βarrestin recruitment and alanine substitution of I284 ECL3 , K291 7.32 and N276 6.55 do not reduce the paltusotine induced β-arrestin recruitment. The contact of the ligand within the extended binding pocket of SSTR2 is likely to be involved in β-arrestin signal bias modulation, thus designing specific drugs target the core region and with minimum contact with the extended binding pocket would facilitate the development of G proteinbiased ligand of SSTR2.
Furthermore, we tend to find residues involved in bias regulation in the orthosteric site. Interestingly, our results revealed that replacement of F294 7. 35 with alanine nearly impaired the octreotide induced β-arrestin recruitment (Fig. R11a-c), however, the F294 7. 35 A mutant only slightly influenced the β-arrestin signal of SSTR2 in response to paltusotine (Fig. R11d-f). In addition, structural comparison indicated that the side chain of F294 is closer to octreotide than paltusotine, despite of different rotameric displacement in the two structures. Combined with structural observation, our results of cAMP inhibition as well as β-arrestin recruitment assays enabled us to speculate that F294 7. 35 should be engaged in modulation of β-arrestin recruitment. Collectively, the understanding of the bias properties of SSTR2 would help pharmacologists to design or optimize efficacious small molecules with biased signaling. Statistical differences between wild-type and mutants were determined by one way of variance ANOVA with Dunnett's test. *P < 0.033, ***p <0.01 n.s., not significant, n.d., not detected. Data represent mean ± SEM from three independent experiments. d-f: The effects of SSTR2 I284 ECL3 , K291 7.32 , N276 6.55 A and F294 7. 35 mutations on β-arrestin recruitment(d) and cAMP inhibition (e) induced by paltusotine. f. Bias factors of these mutants.
Statistical differences between wild-type and mutants were determined by one way of variance ANOVA with Dunnett's test. *P < 0.033, ***p <0.01 n.s., not significant, n.d., not detected. Data represent mean ± SEM from three independent experiments.
Other minor suggestions include: 1. In line 29, the authors claim that "drug resistance occurs in a subset of patients, which may be correlated with SSTR subtype selectivity or cell surface expression." I understand the correlation between 'drug resistance' and 'cell surface expression'. But I don't quite understand why 'subtype selectivity' correlates with 'drug resistance'.
Response: We gratefully appreciate the reviewer for pointing out the correlation between drug resistance with subtype selectivity or cell surface expression. We apologize for this ambiguous description about the correlation between them. Subtype selectivity of drug discovery for specific SSTR member is actually valuable therapeutic strategy since five SSTRs are involved in divergent physiological functions, and some progresses have been made in selective SST analogs or small molecules with better pharmacological profile. We didn't find a clue of the correlation of drug resistance with subtype selectivity from previous literatures, therefore, we revised the sentence in the revised manuscript, line 29.
2. The confocal fluorescence microscopy images are confusing to me. For example, in Fig. 6c and 6d, it looks like paltusotine induces as much internalization as octreotide does in WT SSTR2. While in Fig. 6c, it looks like octreotide induces as much internalization for N276 6.55 A and F294 7.35 A mutants compared to WT SSTR2. These results are not consistent with the main conclusion of the manuscript.
Response: We thank the reviewer for pointing out this issue. We checked the raw data carefully and found that we placed the confocal image in the wrong order in the previous version of manuscript and we have corrected it (Fig. R12a, b). We apologize for this confusing figure presentation. In the revised manuscript, to make sure of the authenticity of the data, we repeated the confocal fluorescence microscopy experiments to support our standpoint. As is shown in Fig. R12c and d, in consistent with our previous data, the results revealed that both N276 6.55 A and F294 7.35 A mutations showed diminished internalization of SSTR2 when treated with octreotide, while only slightly influenced the internalization under paltusotine treatment.